Natural history of epilepsy in argininosuccinic aciduria provides new insights into pathophysiology: A retrospective international study.

OBJECTIVE: Argininosuccinate lyase (ASL) is integral to the urea cycle, which enables nitrogen wasting and biosynthesis of arginine, a precursor of nitric oxide. Inherited ASL deficiency causes argininosuccinic aciduria, the second most common urea cycle defect and an inherited model of systemic nitric oxide deficiency. Patients present with developmental delay, epilepsy, and movement disorder. Here we aim to characterize epilepsy, a common and neurodebilitating comorbidity in argininosuccinic aciduria. METHODS: We conducted a retrospective study in seven tertiary metabolic centers in the UK, Italy, and Canada from 2020 to 2022, to assess the phenotype of epilepsy in argininosuccinic aciduria and correlate it with clinical, biochemical, radiological, and electroencephalographic data. RESULTS: Thirty-seven patients, 1-31 years of age, were included. Twenty-two patients (60%) presented with epilepsy. The median age at epilepsy onset was 24 months. Generalized tonic-clonic and focal seizures were most common in early-onset patients, whereas atypical absences were predominant in late-onset patients. Seventeen patients (77%) required antiseizure medications and six (27%) had pharmacoresistant epilepsy. Patients with epilepsy presented with a severe neurodebilitating disease with higher rates of speech delay (p = .04) and autism spectrum disorders (p = .01) and more frequent arginine supplementation (p = .01) compared to patients without epilepsy. Neonatal seizures were not associated with a higher risk of developing epilepsy. Biomarkers of ureagenesis did not differ between epileptic and non-epileptic patients. Epilepsy onset in early infancy (p = .05) and electroencephalographic background asymmetry (p = .0007) were significant predictors of partially controlled or refractory epilepsy. SIGNIFICANCE: Epilepsy in argininosuccinic aciduria is frequent, polymorphic, and associated with more frequent neurodevelopmental comorbidities. We identified prognostic factors for pharmacoresistance in epilepsy. This study does not support defective ureagenesis as prominent in the pathophysiology of epilepsy but suggests a role of central dopamine deficiency. A role of arginine in epileptogenesis was not supported and warrants further studies to assess the potential arginine neurotoxicity in argininosuccinic aciduria.

Postauthorization safety study of betaine anhydrous.

Patient registries for rare diseases enable systematic data collection and can also be used to facilitate postauthorization safety studies (PASS) for orphan drugs. This study evaluates the PASS for betaine anhydrous (Cystadane), conducted as public private partnership (PPP) between the European network and registry for homocystinurias and methylation defects and the marketing authorization holder (MAH). Data were prospectively collected, 2013-2016, in a noninterventional, international, multicenter, registry study. Putative adverse and severe adverse events were reported to the MAH's pharmacovigilance. In total, 130 individuals with vitamin B6 nonresponsive (N = 54) and partially responsive (N = 7) cystathionine beta-synthase (CBS) deficiency, as well as 5,10-methylenetetrahydrofolate reductase (MTHFR; N = 21) deficiency and cobalamin C (N = 48) disease were included. Median (range) duration of treatment with betaine anhydrous was 6.8 (0-9.8) years. The prescribed betaine dose exceeded the recommended maximum (6 g/day) in 49% of individuals older than 10 years because of continued dose adaptation to weight; however, with disease-specific differences (minimum: 31% in B6 nonresponsive CBS deficiency, maximum: 67% in MTHFR deficiency). Despite dose escalation no new or potential risk was identified. Combined disease-specific treatment decreased mean ± SD total plasma homocysteine concentrations from 203 ± 116 to 81 ± 51 µmol/L (p < 0.0001), except in MTHFR deficiency. Recommendations for betaine anhydrous dosage were revised for individuals ≥ 10 years. PPPs between MAH and international scientific consortia can be considered a reliable model for implementing a PASS, reutilizing well-established structures and avoiding data duplication and fragmentation.

Neurocognitive outcome and mental health in children with tyrosinemia type 1 and phenylketonuria: A comparison between two genetic disorders affecting the same metabolic pathway.

Tyrosinemia type 1 (TT1) and phenylketonuria (PKU) are both inborn errors of phenylalanine-tyrosine metabolism. Neurocognitive and behavioral outcomes have always featured in PKU research but received less attention in TT1 research. This study aimed to investigate and compare neurocognitive, behavioral, and social outcomes of treated TT1 and PKU patients. We included 33 TT1 patients (mean age 11.24 years; 16 male), 31 PKU patients (mean age 10.84; 14 male), and 58 age- and gender-matched healthy controls (mean age 10.82 years; 29 male). IQ (Wechsler-subtests), executive functioning (the Behavioral Rating Inventory of Executive Functioning), mental health (the Achenbach-scales), and social functioning (the Social Skills Rating System) were assessed. Results of TT1 patients, PKU patients, and healthy controls were compared using Kruskal-Wallis tests with post-hoc Mann-Whitney U tests. TT1 patients showed a lower IQ and poorer executive functioning, mental health, and social functioning compared to healthy controls and PKU patients. PKU patients did not differ from healthy controls regarding these outcome measures. Relatively poor outcomes for TT1 patients were particularly evident for verbal IQ, BRIEF dimensions "working memory", "plan and organize" and "monitor", ASEBA dimensions "social problems" and "attention problems", and for the SSRS "assertiveness" scale (all p values <0.001). To conclude, TT1 patients showed cognitive impairments on all domains studied, and appeared to be significantly more affected than PKU patients. More attention should be paid to investigating and monitoring neurocognitive outcome in TT1 and research should focus on explaining the underlying pathophysiological mechanism.

Instability of the mitochondrial alanyl-tRNA synthetase underlies fatal infantile-onset cardiomyopathy.

Recessively inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA synthetase (mt-AlaRS) were first described in patients presenting with fatal infantile cardiomyopathy and multiple oxidative phosphorylation defects. To date, all described patients with AARS2-related fatal infantile cardiomyopathy are united by either a homozygous or compound heterozygous c.1774C>T (p.Arg592Trp) missense founder mutation that is absent in patients with other AARS2-related phenotypes. We describe the clinical, biochemical and molecular investigations of two unrelated boys presenting with fatal infantile cardiomyopathy, lactic acidosis and respiratory failure. Oxidative histochemistry showed cytochrome c oxidase-deficient fibres in skeletal and cardiac muscle. Biochemical studies showed markedly decreased activities of mitochondrial respiratory chain complexes I and IV with a mild decrease of complex III activity in skeletal and cardiac muscle. Using next-generation sequencing, we identified a c.1738C>T (p.Arg580Trp) AARS2 variant shared by both patients that was in trans with a loss-of-function heterozygous AARS2 variant; a c.1008dupT (p.Asp337*) nonsense variant or an intragenic deletion encompassing AARS2 exons 5-7. Interestingly, our patients did not harbour the p.Arg592Trp AARS2 founder mutation. In silico modelling of the p.Arg580Trp substitution suggested a deleterious impact on protein stability and folding. We confirmed markedly decreased mt-AlaRS protein levels in patient fibroblasts, skeletal and cardiac muscle, although mitochondrial protein synthesis defects were confined to skeletal and cardiac muscle. In vitro data showed that the p.Arg580Trp variant had a minimal effect on activation, aminoacylation or misaminoacylation activities relative to wild-type mt-AlaRS, demonstrating that instability of mt-AlaRS is the biological mechanism underlying the fatal cardiomyopathy phenotype in our patients.

Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder.

ATP synthase, H+ transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F1FO ATP synthase and subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation.

Cystathionine ß-synthase deficiency in the E-HOD registry-part I: pyridoxine responsiveness as a determinant of biochemical and clinical phenotype at diagnosis.

Cystathionine ß-synthase (CBS) deficiency has a wide clinical spectrum, ranging from neurodevelopmental problems, lens dislocation and marfanoid features in early childhood to adult onset disease with predominantly thromboembolic complications. We have analysed clinical and laboratory data at the time of diagnosis in 328 patients with CBS deficiency from the E-HOD (European network and registry for Homocystinurias and methylation Defects) registry. We developed comprehensive criteria to classify patients into four groups of pyridoxine responsivity: non-responders (NR), partial, full and extreme responders (PR, FR and ER, respectively). All groups showed overlapping concentrations of plasma total homocysteine while pyridoxine responsiveness inversely correlated with plasma/serum methionine concentrations. The FR and ER groups had a later age of onset and diagnosis and a longer diagnostic delay than NR and PR patients. Lens dislocation was common in all groups except ER but the age of dislocation increased with increasing responsiveness. Developmental delay was commonest in the NR group while no ER patient had cognitive impairment. Thromboembolism was the commonest presenting feature in ER patients, whereas it was least likely at presentation in the NR group. This probably is due to the differences in ages at presentation: all groups had a similar number of thromboembolic events per 1000 patient-years. Clinical severity of CBS deficiency depends on the degree of pyridoxine responsiveness. Therefore, a standardised pyridoxine-responsiveness test in newly diagnosed patients and a critical review of previous assessments is indispensable to ensure adequate therapy and to prevent or reduce long-term complications.

Efficacy and safety of D,L-3-hydroxybutyrate (D,L-3-HB) treatment in multiple acyl-CoA dehydrogenase deficiency.

PURPOSE: Multiple acyl-CoA dehydrogenase deficiency (MADD) is a life-threatening, ultrarare inborn error of metabolism. Case reports described successful D,L-3-hydroxybutyrate (D,L-3-HB) treatment in severely affected MADD patients, but systematic data on efficacy and safety is lacking. METHODS: A systematic literature review and an international, retrospective cohort study on clinical presentation, D,L-3-HB treatment method, and outcome in MADD(-like) patients. RESULTS: Our study summarizes 23 MADD(-like) patients, including 14 new cases. Median age at clinical onset was two months (interquartile range [IQR]: 8 months). Median age at starting D,L-3-HB was seven months (IQR: 4.5 years). D,L-3-HB doses ranged between 100 and 2600 mg/kg/day. Clinical improvement was reported in 16 patients (70%) for cardiomyopathy, leukodystrophy, liver symptoms, muscle symptoms, and/or respiratory failure. D,L-3-HB appeared not effective for neuropathy. Survival appeared longer upon D,L-3-HB compared with historical controls. Median time until first clinical improvement was one month, and ranged up to six months. Reported side effects included abdominal pain, constipation, dehydration, diarrhea, and vomiting/nausea. Median D,L-3-HB treatment duration was two years (IQR: 6 years). D,L-3-HB treatment was discontinued in 12 patients (52%). CONCLUSION: The strength of the current study is the international pooling of data demonstrating that D,L-3-HB treatment can be effective and safe in MADD(-like) patients.

Recessive Mutations in TRMT10C Cause Defects in Mitochondrial RNA Processing and Multiple Respiratory Chain Deficiencies.

Mitochondrial disorders are clinically and genetically diverse, with mutations in mitochondrial or nuclear genes able to cause defects in mitochondrial gene expression. Recently, mutations in several genes encoding factors involved in mt-tRNA processing have been identified to cause mitochondrial disease. Using whole-exome sequencing, we identified mutations in TRMT10C (encoding the mitochondrial RNase P protein 1 [MRPP1]) in two unrelated individuals who presented at birth with lactic acidosis, hypotonia, feeding difficulties, and deafness. Both individuals died at 5 months after respiratory failure. MRPP1, along with MRPP2 and MRPP3, form the mitochondrial ribonuclease P (mt-RNase P) complex that cleaves the 5' ends of mt-tRNAs from polycistronic precursor transcripts. Additionally, a stable complex of MRPP1 and MRPP2 has m(1)R9 methyltransferase activity, which methylates mt-tRNAs at position 9 and is vital for folding mt-tRNAs into their correct tertiary structures. Analyses of fibroblasts from affected individuals harboring TRMT10C missense variants revealed decreased protein levels of MRPP1 and an increase in mt-RNA precursors indicative of impaired mt-RNA processing and defective mitochondrial protein synthesis. The pathogenicity of the detected variants-compound heterozygous c.542G>T (p.Arg181Leu) and c.814A>G (p.Thr272Ala) changes in subject 1 and a homozygous c.542G>T (p.Arg181Leu) variant in subject 2-was validated by the functional rescue of mt-RNA processing and mitochondrial protein synthesis defects after lentiviral transduction of wild-type TRMT10C. Our study suggests that these variants affect MRPP1 protein stability and mt-tRNA processing without affecting m(1)R9 methyltransferase activity, identifying mutations in TRMT10C as a cause of mitochondrial disease and highlighting the importance of RNA processing for correct mitochondrial function.

Phenotype, treatment practice and outcome in the cobalamin-dependent remethylation disorders and MTHFR deficiency: Data from the E-HOD registry.

AIM: To explore the clinical presentation, course, treatment and impact of early treatment in patients with remethylation disorders from the European Network and Registry for Homocystinurias and Methylation Defects (E-HOD) international web-based registry. RESULTS: This review comprises 238 patients (cobalamin C defect n = 161; methylenetetrahydrofolate reductase deficiency n = 50; cobalamin G defect n = 11; cobalamin E defect n = 10; cobalamin D defect n = 5; and cobalamin J defect n = 1) from 47 centres for whom the E-HOD registry includes, as a minimum, data on medical history and enrolment visit. The duration of observation was 127 patient years. In 181 clinically diagnosed patients, the median age at presentation was 30 days (range 1 day to 42 years) and the median age at diagnosis was 3.7 months (range 3 days to 56 years). Seventy-five percent of pre-clinically diagnosed patients with cobalamin C disease became symptomatic within the first 15 days of life. Total homocysteine (tHcy), amino acids and urinary methylmalonic acid (MMA) were the most frequently assessed disease markers; confirmatory diagnostics were mainly molecular genetic studies. Remethylation disorders are multisystem diseases dominated by neurological and eye disease and failure to thrive. In this cohort, mortality, thromboembolic, psychiatric and renal disease were rarer than reported elsewhere. Early treatment correlates with lower overall morbidity but is less effective in preventing eye disease and cognitive impairment. The wide variation in treatment hampers the evaluation of particular therapeutic modalities. CONCLUSION: Treatment improves the clinical course of remethylation disorders and reduces morbidity, especially if started early, but neurocognitive and eye symptoms are less responsive. Current treatment is highly variable. This study has the inevitable limitations of a retrospective, registry-based design.

Newborn screening for homocystinurias: Recent recommendations versus current practice.

PURPOSE: To assess how the current practice of newborn screening (NBS) for homocystinurias compares with published recommendations. METHODS: Twenty-two of 32 NBS programmes from 18 countries screened for at least one form of homocystinuria. Centres provided pseudonymised NBS data from patients with cystathionine beta-synthase deficiency (CBSD, n = 19), methionine adenosyltransferase I/III deficiency (MATI/IIID, n = 28), combined remethylation disorder (cRMD, n = 56) and isolated remethylation disorder (iRMD), including methylenetetrahydrofolate reductase deficiency (MTHFRD) (n = 8). Markers and decision limits were converted to multiples of the median (MoM) to allow comparison between centres. RESULTS: NBS programmes, algorithms and decision limits varied considerably. Only nine centres used the recommended second-tier marker total homocysteine (tHcy). The median decision limits of all centres were ≥ 2.35 for high and ≤ 0.44 MoM for low methionine, ≥ 1.95 for high and ≤ 0.47 MoM for low methionine/phenylalanine, ≥ 2.54 for high propionylcarnitine and ≥ 2.78 MoM for propionylcarnitine/acetylcarnitine. These decision limits alone had a 100%, 100%, 86% and 84% sensitivity for the detection of CBSD, MATI/IIID, iRMD and cRMD, respectively, but failed to detect six individuals with cRMD. To enhance sensitivity and decrease second-tier testing costs, we further adapted these decision limits using the data of 15 000 healthy newborns. CONCLUSIONS: Due to the favorable outcome of early treated patients, NBS for homocystinurias is recommended. To improve NBS, decision limits should be revised considering the population median. Relevant markers should be combined; use of the postanalytical tools offered by the CLIR project (Collaborative Laboratory Integrated Reports, which considers, for example, birth weight and gestational age) is recommended. tHcy and methylmalonic acid should be implemented as second-tier markers.

The genotypic and phenotypic spectrum of MTO1 deficiency.

BACKGROUND: Mitochondrial diseases, a group of multi-systemic disorders often characterized by tissue-specific phenotypes, are usually progressive and fatal disorders resulting from defects in oxidative phosphorylation. MTO1 (Mitochondrial tRNA Translation Optimization 1), an evolutionarily conserved protein expressed in high-energy demand tissues has been linked to human early-onset combined oxidative phosphorylation deficiency associated with hypertrophic cardiomyopathy, often referred to as combined oxidative phosphorylation deficiency-10 (COXPD10). MATERIAL AND METHODS: Thirty five cases of MTO1 deficiency were identified and reviewed through international collaboration. The cases of two female siblings, who presented at 1 and 2years of life with seizures, global developmental delay, hypotonia, elevated lactate and complex I and IV deficiency on muscle biopsy but without cardiomyopathy, are presented in detail. RESULTS: For the description of phenotypic features, the denominator varies as the literature was insufficient to allow for complete ascertainment of all data for the 35 cases. An extensive review of all known MTO1 deficiency cases revealed the most common features at presentation to be lactic acidosis (LA) (21/34; 62% cases) and hypertrophic cardiomyopathy (15/34; 44% cases). Eventually lactic acidosis and hypertrophic cardiomyopathy are described in 35/35 (100%) and 27/34 (79%) of patients with MTO1 deficiency, respectively; with global developmental delay/intellectual disability present in 28/29 (97%), feeding difficulties in 17/35 (49%), failure to thrive in 12/35 (34%), seizures in 12/35 (34%), optic atrophy in 11/21 (52%) and ataxia in 7/34 (21%). There are 19 different pathogenic MTO1 variants identified in these 35 cases: one splice-site, 3 frameshift and 15 missense variants. None have bi-allelic variants that completely inactivate MTO1; however, patients where one variant is truncating (i.e. frameshift) while the second one is a missense appear to have a more severe, even fatal, phenotype. These data suggest that complete loss of MTO1 is not viable. A ketogenic diet may have exerted a favourable effect on seizures in 2/5 patients. CONCLUSION: MTO1 deficiency is lethal in some but not all cases, and a genotype-phenotype relation is suggested. Aside from lactic acidosis and cardiomyopathy, developmental delay and other phenotypic features affecting multiple organ systems are often present in these patients, suggesting a broader spectrum than hitherto reported. The diagnosis should be suspected on clinical features and the presence of markers of mitochondrial dysfunction in body fluids, especially low residual complex I, III and IV activity in muscle. Molecular confirmation is required and targeted genomic testing may be the most efficient approach. Although subjective clinical improvement was observed in a small number of patients on therapies such as ketogenic diet and dichloroacetate, no evidence-based effective therapy exists.

Novel GFM2 variants associated with early-onset neurological presentations of mitochondrial disease and impaired expression of OXPHOS subunits.

Mitochondrial diseases are characterised by clinical, molecular and functional heterogeneity, reflecting their bi-genomic control. The nuclear gene GFM2 encodes mtEFG2, a protein with an essential role during the termination stage of mitochondrial translation. We present here two unrelated patients harbouring different and previously unreported compound heterozygous (c.569G>A, p.(Arg190Gln); c.636delA, p.(Glu213Argfs*3)) and homozygous (c.275A>C, p.(Tyr92Ser)) recessive variants in GFM2 identified by whole exome sequencing (WES) together with histochemical and biochemical findings to support the diagnoses of pathological GFM2 variants in each case. Both patients presented similarly in early childhood with global developmental delay, raised CSF lactate and abnormalities on cranial MRI. Sanger sequencing of familial samples confirmed the segregation of bi-allelic GFM2 variants with disease, while investigations into steady-state mitochondrial protein levels revealed respiratory chain subunit defects and loss of mtEFG2 protein in muscle. These data demonstrate the effects of defective mtEFG2 function, caused by previously unreported variants, confirming pathogenicity and expanding the clinical phenotypes associated with GFM2 variants.

Expanding the phenotype in argininosuccinic aciduria: need for new therapies.

OBJECTIVES: This UK-wide study defines the natural history of argininosuccinic aciduria and compares long-term neurological outcomes in patients presenting clinically or treated prospectively from birth with ammonia-lowering drugs. METHODS: Retrospective analysis of medical records prior to March 2013, then prospective analysis until December 2015. Blinded review of brain MRIs. ASL genotyping. RESULTS: Fifty-six patients were defined as early-onset (n = 23) if symptomatic < 28 days of age, late-onset (n = 23) if symptomatic later, or selectively screened perinatally due to a familial proband (n = 10). The median follow-up was 12.4 years (range 0-53). Long-term outcomes in all groups showed a similar neurological phenotype including developmental delay (48/52), epilepsy (24/52), ataxia (9/52), myopathy-like symptoms (6/52) and abnormal neuroimaging (12/21). Neuroimaging findings included parenchymal infarcts (4/21), focal white matter hyperintensity (4/21), cortical or cerebral atrophy (4/21), nodular heterotopia (2/21) and reduced creatine levels in white matter (4/4). 4/21 adult patients went to mainstream school without the need of additional educational support and 1/21 lives independently. Early-onset patients had more severe involvement of visceral organs including liver, kidney and gut. All early-onset and half of late-onset patients presented with hyperammonaemia. Screened patients had normal ammonia at birth and received treatment preventing severe hyperammonaemia. ASL was sequenced (n = 19) and 20 mutations were found. Plasma argininosuccinate was higher in early-onset compared to late-onset patients. CONCLUSIONS: Our study further defines the natural history of argininosuccinic aciduria and genotype-phenotype correlations. The neurological phenotype does not correlate with the severity of hyperammonaemia and plasma argininosuccinic acid levels. The disturbance in nitric oxide synthesis may be a contributor to the neurological disease. Clinical trials providing nitric oxide to the brain merit consideration.

Guidelines for the diagnosis and management of cystathionine beta-synthase deficiency.

Cystathionine beta-synthase (CBS) deficiency is a rare inherited disorder in the methionine catabolic pathway, in which the impaired synthesis of cystathionine leads to accumulation of homocysteine. Patients can present to many different specialists and diagnosis is often delayed. Severely affected patients usually present in childhood with ectopia lentis, learning difficulties and skeletal abnormalities. These patients generally require treatment with a low-methionine diet and/or betaine. In contrast, mildly affected patients are likely to present as adults with thromboembolism and to respond to treatment with pyridoxine. In this article, we present recommendations for the diagnosis and management of CBS deficiency, based on a systematic review of the literature. Unfortunately, the quality of the evidence is poor, as it often is for rare diseases. We strongly recommend measuring the plasma total homocysteine concentrations in any patient whose clinical features suggest the diagnosis. Our recommendations may help to standardise testing for pyridoxine responsiveness. Current evidence suggests that patients are unlikely to develop complications if the plasma total homocysteine concentration is maintained below 120 µmol/L. Nevertheless, we recommend keeping the concentration below 100 µmol/L because levels fluctuate and the complications associated with high levels are so serious.

A recurrent mitochondrial p.Trp22Arg NDUFB3 variant causes a distinctive facial appearance, short stature and a mild biochemical and clinical phenotype.

BACKGROUND: Isolated Complex I deficiency is the most common paediatric mitochondrial disease presentation, associated with poor prognosis and high mortality. Complex I comprises 44 structural subunits with at least 10 ancillary proteins; mutations in 29 of these have so far been associated with mitochondrial disease but there are limited genotype-phenotype correlations to guide clinicians to the correct genetic diagnosis. METHODS: Patients were analysed by whole-exome sequencing, targeted capture or candidate gene sequencing. Clinical phenotyping of affected individuals was performed. RESULTS: We identified a cohort of 10 patients from 8 families (7 families are of unrelated Irish ancestry) all of whom have short stature (<9th centile) and similar facial features including a prominent forehead, smooth philtrum and deep-set eyes associated with a recurrent homozygous c.64T>C, p.Trp22Arg NDUFB3 variant. Two sibs presented with primary short stature without obvious metabolic dysfunction. Analysis of skeletal muscle from three patients confirmed a defect in Complex I assembly. CONCLUSIONS: Our report highlights that the long-term prognosis related to the p.Trp22Arg NDUFB3 mutation can be good, even for some patients presenting in acute metabolic crisis with evidence of an isolated Complex I deficiency in muscle. Recognition of the distinctive facial features-particularly when associated with markers of mitochondrial dysfunction and/or Irish ancestry-should suggest screening for the p.Trp22Arg NDUFB3 mutation to establish a genetic diagnosis, circumventing the requirement of muscle biopsy to direct genetic investigations.

The clinical, biochemical and genetic features associated with RMND1-related mitochondrial disease.

BACKGROUND: Mutations in the RMND1 (Required for Meiotic Nuclear Division protein 1) gene have recently been linked to infantile onset mitochondrial disease characterised by multiple mitochondrial respiratory chain defects. METHODS: We summarised the clinical, biochemical and molecular genetic investigation of an international cohort of affected individuals with RMND1 mutations. In addition, we reviewed all the previously published cases to determine the genotype-phenotype correlates and performed survival analysis to identify prognostic factors. RESULTS: We identified 14 new cases from 11 pedigrees that harbour recessive RMND1 mutations, including 6 novel variants: c.533C>A, p.(Thr178Lys); c.565C>T, p.(Gln189*); c.631G>A, p.(Val211Met); c.1303C>T, p.(Leu435Phe); c.830+1G>A and c.1317+1G>T. Together with all previously published cases (n=32), we show that congenital sensorineural deafness, hypotonia, developmental delay and lactic acidaemia are common clinical manifestations with disease onset under 2 years. Renal involvement is more prevalent than seizures (66% vs 44%). In addition, median survival time was longer in patients with renal involvement compared with those without renal disease (6 years vs 8 months, p=0.009). The neurological phenotype also appears milder in patients with renal involvement. CONCLUSIONS: The clinical phenotypes and prognosis associated with RMND1 mutations are more heterogeneous than that were initially described. Regular monitoring of kidney function is imperative in the clinical practice in light of nephropathy being present in over 60% of cases. Furthermore, renal replacement therapy should be considered particularly in those patients with mild neurological manifestation as shown in our study that four recipients of kidney transplant demonstrate good clinical outcome to date.

Clinical pattern, mutations and in vitro residual activity in 33 patients with severe 5, 10 methylenetetrahydrofolate reductase (MTHFR) deficiency.

BACKGROUND: Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is a rare inborn defect disturbing the remethylation of homocysteine to methionine (<200 reported cases). This retrospective study evaluates clinical, biochemical genetic and in vitro enzymatic data in a cohort of 33 patients. METHODS: Clinical, biochemical and treatment data was obtained from physicians by using a questionnaire. MTHFR activity was measured in primary fibroblasts; genomic DNA was extracted from cultured fibroblasts. RESULTS: Thirty-three patients (mean age at follow-up 11.4 years; four deceased; median age at first presentation 5 weeks; 17 females) were included. Patients with very low (<1.5%) mean control values of enzyme activity (n = 14) presented earlier and with a pattern of feeding problems, encephalopathy, muscular hypotonia, neurocognitive impairment, apnoea, hydrocephalus, microcephaly and epilepsy. Patients with higher (>1.7-34.8%) residual enzyme activity had mainly psychiatric symptoms, mental retardation, myelopathy, ataxia and spasticity. Treatment with various combinations of betaine, methionine, folate and cobalamin improved the biochemical and clinical phenotype. During the disease course, patients with very low enzyme activity showed a progression of feeding problems, neurological symptoms, mental retardation, and psychiatric disease while in patients with higher residual enzyme activity, myelopathy, ataxia and spasticity increased. All other symptoms remained stable or improved in both groups upon treatment as did brain imaging in some cases. No clear genotype-phenotype correlation was obvious. DISCUSSION: MTHFR deficiency is a severe disease primarily affecting the central nervous system. Age at presentation and clinical pattern are correlated with residual enzyme activity. Treatment alleviates biochemical abnormalities and clinical symptoms partially.

LRPPRC mutations cause early-onset multisystem mitochondrial disease outside of the French-Canadian population.

Mitochondrial Complex IV [cytochrome c oxidase (COX)] deficiency is one of the most common respiratory chain defects in humans. The clinical phenotypes associated with COX deficiency include liver disease, cardiomyopathy and Leigh syndrome, a neurodegenerative disorder characterized by bilateral high signal lesions in the brainstem and basal ganglia. COX deficiency can result from mutations affecting many different mitochondrial proteins. The French-Canadian variant of COX-deficient Leigh syndrome is unique to the Saguenay-Lac-Saint-Jean region of Québec and is caused by a founder mutation in the LRPPRC gene. This encodes the leucine-rich pentatricopeptide repeat domain protein (LRPPRC), which is involved in post-transcriptional regulation of mitochondrial gene expression. Here, we present the clinical and molecular characterization of novel, recessive LRPPRC gene mutations, identified using whole exome and candidate gene sequencing. The 10 patients come from seven unrelated families of UK-Caucasian, UK-Pakistani, UK-Indian, Turkish and Iraqi origin. They resemble the French-Canadian Leigh syndrome patients in having intermittent severe lactic acidosis and early-onset neurodevelopmental problems with episodes of deterioration. In addition, many of our patients have had neonatal cardiomyopathy or congenital malformations, most commonly affecting the heart and the brain. All patients who were tested had isolated COX deficiency in skeletal muscle. Functional characterization of patients' fibroblasts and skeletal muscle homogenates showed decreased levels of mutant LRPPRC protein and impaired Complex IV enzyme activity, associated with abnormal COX assembly and reduced steady-state levels of numerous oxidative phosphorylation subunits. We also identified a Complex I assembly defect in skeletal muscle, indicating different roles for LRPPRC in post-transcriptional regulation of mitochondrial mRNAs between tissues. Patient fibroblasts showed decreased steady-state levels of mitochondrial mRNAs, although the length of poly(A) tails of mitochondrial transcripts were unaffected. Our study identifies LRPPRC as an important disease-causing gene in an early-onset, multisystem and neurological mitochondrial disease, which should be considered as a cause of COX deficiency even in patients originating outside of the French-Canadian population.

Copy number variation in the human Y chromosome in the UK population.

We have assessed copy number variation (CNV) in the male-specific part of the human Y chromosome discovered by array comparative genomic hybridization (array-CGH) in 411 apparently healthy UK males, and validated the findings using SNP genotype intensity data available for 149 of them. After manual curation taking account of the complex duplicated structure of Y-chromosomal sequences, we discovered 22 curated CNV events considered validated or likely, mean 0.93 (range 0-4) per individual. 16 of these were novel. Curated CNV events ranged in size from <1 kb to >3 Mb, and in frequency from 1/411 to 107/411. Of the 24 protein-coding genes or gene families tested, nine showed CNV. These included a large duplication encompassing the AMELY and TBL1Y genes that probably has no phenotypic effect, partial deletions of the TSPY cluster and AZFc region that may influence spermatogenesis, and other variants with unknown functional implications, including abundant variation in the number of RBMY genes and/or pseudogenes, and a novel complex duplication of two segments overlapping the AZFa region and including the 3' end of the UTY gene.

Quantitative charge-tags for sterol and oxysterol analysis.

BACKGROUND: Global sterol analysis is challenging owing to the extreme diversity of sterol natural products, the tendency of cholesterol to dominate in abundance over all other sterols, and the structural lack of a strong chromophore or readily ionized functional group. We developed a method to overcome these challenges by using different isotope-labeled versions of the Girard P reagent (GP) as quantitative charge-tags for the LC-MS analysis of sterols including oxysterols. METHODS: Sterols/oxysterols in plasma were extracted in ethanol containing deuterated internal standards, separated by C18 solid-phase extraction, and derivatized with GP, with or without prior oxidation of 3ß-hydroxy to 3-oxo groups. RESULTS: By use of different isotope-labeled GPs, it was possible to analyze in a single LC-MS analysis both sterols/oxysterols that naturally possess a 3-oxo group and those with a 3ß-hydroxy group. Intra- and interassay CVs were <15%, and recoveries for representative oxysterols and cholestenoic acids were 85%-108%. By adopting a multiplex approach to isotope labeling, we analyzed up to 4 different samples in a single run. Using plasma samples, we could demonstrate the diagnosis of inborn errors of metabolism and also the export of oxysterols from brain via the jugular vein. CONCLUSIONS: This method allows the profiling of the widest range of sterols/oxysterols in a single analytical run and can be used to identify inborn errors of cholesterol synthesis and metabolism.